Pomegranate Fruit Cracking during Maturation: From Waste to Valuable Fruits

The pomegranate is an emerging functional food which is nowadays becoming more and more commercially attractive. Each part of this fruit (peels, arils and seeds) has a specific phytocomplex, rich in anti-oxidant and anti-radical compounds. Among these, punicalagin and ellagic acid continue to be widely studied for their numerous beneficial effects on human health (anti-inflammatory effects, anti-diabetes activity, cardio-protection, cancer prevention). Despite their exceptionally valuable composition and high adaptability to different climatic conditions, pomegranate fruits are highly susceptible to splitting during different stages of ripening, so much so that an estimated 65% of the production may be lost. A “zero-kilometer” approach should therefore be adopted to utilize such a valuable product otherwise destined to be downgraded or even incinerated, with a very high environmental impact. The aim of this work is to highlight and compare the compositional differences between whole and split pomegranates belonging to the cultivar Dente di Cavallo, grown in Apulia (Italy), to assess a valuable role for this split fruit usually considered as waste. The arils and peels are subjected to extraction procedures and the extracts analyzed by CIEL*a*b*, HPLC-DAD and HS-SPME/GC-MS. Moreover, an assessment of the inhibitory activity against α-glucosidase, acetylcholinesterase and tyrosinase enzymes has also been applied. The data show a better chemical profile in split fruits (namely 60% more anthocyanin content than intact fruit) with very interesting results in terms of α-glucosidase inhibition. The juices obtained by squeezing are also compared to commercial juices (“Salus Melagrana” and “La Marianna”) processed from the same cultivar and subjected to the same protocol analysis.


Introduction
The nutritional potential of pomegranates as an emerging functional food is currently growing, together with consumer demand, making this fruit an interesting commercial area. In addition to the nutraceutical properties of its different botanical parts, Punica granatum represents a species that is highly adaptable to different climates, arid areas included. It is

Materials
Ethanol, methanol and acetonitrile (HPLC-grade) were obtained from Merck Science Life s.r.l (Milan, Italy). All solvents and chemical standards used in this paper were analytical grade products purchased from Merck Science Life s.r.l (Milan, Italy) and were used without any further purification.

Samples
The pomegranate fruit (Punica granatum L.) cv. "Dente di cavallo", harvested intact and split, and the pomegranate juice "Salus Melagrana" (Eurosalus Italia Srl-Via Francia 6G, Negrar, 37024 (Verona, Italy)) and "Succo La Marianna" were collected or obtained by cold pressing from Fratelli Palmieri, Casalnuovo M.ro (Foggia, Italy) in the Fortore River valley, a natural oasis for the protection of plant and animal biodiversity. The whole fruit (W), separated peels (P) and their squeezed juice (J), each obtained from five different pomegranates, were submitted to different analyses for the phytocomplex characterization. All the experiments were performed in quadruplicate. Both intact and split fruit were harvested to assess any differences in content or biological activity ( Figure 1).

Hydroalcoholic Extraction
W and J, obtained both from the intact fruit and from the split fruit, and P, obtained from the split fruit, were submitted to the hydroalcoholic extraction procedure as previously described by Altieri et al. (2019) [9]. Samples (10 g) from approximately 10 kg were randomly selected from different bulks representative of the whole seasonal harvest, blended and extracted with 40 mL of ethanol:acidified water (5% acetic acid) in 3:1 (v:v) ratio, with stirring for 1 h at room temperature in the dark. The extraction mixture was decanted, filtered and evaporated at 40 °C under vacuum and stored at 4 °C until the analyses were performed (samples of HAW, HAP and HAJ).

Materials
Ethanol, methanol and acetonitrile (HPLC-grade) were obtained from Merck Science Life s.r.l (Milan, Italy). All solvents and chemical standards used in this paper were analytical grade products purchased from Merck Science Life s.r.l (Milan, Italy) and were used without any further purification.

Samples
The pomegranate fruit (Punica granatum L.) cv. "Dente di cavallo", harvested intact and split, and the pomegranate juice "Salus Melagrana" (Eurosalus Italia Srl-Via Francia 6G, Negrar, 37024 (Verona, Italy)) and "Succo La Marianna" were collected or obtained by cold pressing from Fratelli Palmieri, Casalnuovo M.ro (Foggia, Italy) in the Fortore River valley, a natural oasis for the protection of plant and animal biodiversity. The whole fruit (W), separated peels (P) and their squeezed juice (J), each obtained from five different pomegranates, were submitted to different analyses for the phytocomplex characterization. All the experiments were performed in quadruplicate. Both intact and split fruit were harvested to assess any differences in content or biological activity ( Figure 1).

Hydroalcoholic Extraction
W and J, obtained both from the intact fruit and from the split fruit, and P, obtained from the split fruit, were submitted to the hydroalcoholic extraction procedure as previously described by Altieri et al. (2019) [9]. Samples (10 g) from approximately 10 kg were randomly selected from different bulks representative of the whole seasonal harvest, blended and extracted with 40 mL of ethanol:acidified water (5% acetic acid) in 3:1 (v:v) ratio, with stirring for 1 h at room temperature in the dark. The extraction mixture was decanted, filtered and evaporated at 40 • C under vacuum and stored at 4 • C until the analyses were performed (samples of HA W , HA P and HA J ).

Anthocyanin Extraction
HA W , HA P and HA J were subjected to solid phase extraction (SPE) for the purification and quantification of anthocyanin. The extraction was performed using a Discovery ®  [10], with substantial modifications. The column was conditioned beforehand with 5 mL of ethyl acetate, 5 mL of methanol (5% CH 3 COOH v/v) and, finally, with 2 mL of H 2 O (5% CH 3 COOH v/v). Then, about 100 mg/mL of the samples was loaded into the column. The column was washed with 6 mL of H 2 O (5% CH 3 COOH v/v) and 12 mL of ethyl acetate, which were subsequently discarded. Finally, the anthocyanin fraction was eluted with 4 mL of methanol (5% CH 3 COOH v/v). The obtained fractions were concentrated under reduced pressure at a controlled temperature of 40 • C, weighed and stored at 4 • C until HPLC-DAD analyses were performed (split and intact samples of HA WA and HA JA ).

Colorimetric Analysis and Accelerated Test of Food Shelf-Life
W and J, HA w , HA P and HA J , and the commercial juices "Salus Melagrana" (J S ) and "Succo La Marianna" (J L ) were submitted to colorimetric CIEL*a*b* analysis with a colorimeter X-Rite MetaVue TM® equipped with a full-spectrum LED illuminant and an observer angle of 45 • /0 • imaging spectrophotometer. The analyses were conducted according to Recinella et al. (2021) [11]. The analyses of the juices J S and J L were performed at the time of delivery (t • ) and weekly for five weeks, keeping the samples in the darkness at 37 ± 2 • C.

HPLC-DAD Analysis
The dried extracts taken from the intact and split samples HA w , HA P and HA J and the SPE extracts HA WA and HA JA were weighed and dissolved in a known volume of hydroalcoholic solution (5 mg/mL). The resulting solutions and the commercial juices, such as J S and J L , were filtered with a Millex ® -LG filter (Low Protein Binding Hydrophilic PTFE 0.20 µM Membrane) (Merck Science Life, S.r.l, Milan, Italy), injected and analyzed with an HPLC-DAD (Perkin Elmer, Milan, Italy), equipped with an LC Series 200 pump, a Series 200 DAD, and a Series 200 autosampler, including Perkin Elmer TotalChrom software for data tracking. Analyses were performed on HA w , HA P , HA J , J S and J L at 280 nm for the identification of gallic acid and at 360 nm for the identification of the ellagitannin profile. HA WA and HA JA were analyzed at 520 nm for the identification of anthocyanins. A Luna RP-18, 3 µm column was used, with a linear gradient consisting of acetonitrile and acidified water (5% formic acid), from 100% to 15% aqueous phase in 60 min, at a flow rate of 1.0 mL/min. Calibration curves were expressed in µg/mL and were constructed for gallic acid (y = 15.51x + 37.06; R 2 0.9987), punicalagin (α + β anomers) (y = 3.83x − 49.95; R 2 0.9998), ellagic acid (y = 16.86x + 1.22; R 2 0.9994) and cyanidin-3-O-rutinoside (y = 16.58x + 34.53; R 2 0.9987). Extraction yields of anthocyanins, though quantified on SPE extracts, were finally expressed in relation to the hydroalcoholic extracts to compare the differently obtained data.

HS-SPME/GC-MS Analysis
Dried HA w and HA J samples (0.3 mg), taken from both the split and intact fruit, and HA P split were introduced in 4 mL vials and allowed to equilibrate for 20 min in a thermostat bath set at 80 • C. The equilibration step was followed by the exposure of the CAR-DVB-PDMS fiber to the headspace of the vial for 20 min at 80 • C. Finally, the analytes were allowed to desorb from the fiber exposed into the inlet of an Agilent Technologies 6850 gas chromatograph, coupled with an Agilent Technologies 5975 mass spectrometer, for 0.5 min. The following gas chromatographic layout was used: column, HP-5MS (30 m × 0.25 mm inner diameter, film thickness 0.25 µm); inlet temperature, 260 • C; injection mode, splitless (the split vent was opened after 0.5 min and the split ratio set at the 20/1 value); flow rate of the helium carrier gas (99.995% purity), 1.0 mL/min; oven temperature starting from 40 • C, after 5 min raised to 200 • C at 5 • C/min, and kept at this final temperature for 60 min. Mass spectrometry parameters were set as follows: EI energy, 70 eV; source temperature, 230 • C; quadrupole temperature, 150 • C; the mass scan was carried out over the 50-350 m/z range. The two-level identification of the eluted compounds started from comparing the experimental EI spectra with those collected in both commercial (FFNSC 3) and free databases (NIST 11, Flavor2). The Kovats index (KI) was used as a second parameter to confirm the MS-based identification of the analytes. KIs were measured using a mixture of n-alkanes (C7-C40) in the same chromatographic set-up, and then compared with values reported in the FFNSC 3 and NIST 11 databases. Chromatographic peaks with a S/N ratio above 3 were manually integrated without any further modification.

Enzyme Inhibitory Activity
The samples were subjected to enzyme inhibitory assays against three fundamental enzymes with implications in human pathologies: α-glucosidase, acetylcholinesterase and tyrosinase, using in vitro assays. All the results were expressed in terms of IC 50 (µg/mL), considering a dilution in the microplate, and not the original vial dilution. The percentages of inhibition (I, expressed as %) for every enzyme inhibition assay were calculated using the formula below: where A control is the absorbance of the control solution and A sample is the absorbance of the sample, against blanks. Inhibitory activity against α-glucosidase was assessed using a previously described protocol. Then, 50 µL of different concentrations of the same extract were mixed with 50 µL of α-glucosidase (in a pH 6.8 phosphate buffer solution, PBS). After adding 50 µL of the substrate (4-nitrophenyl-β-D-glucopyranoside PNPG, 10 mM in PBS), the reaction mix was incubated for 5 min at 37 • C, and the absorbance was read at 405 nm. The same protocol was applied for acarbose as positive control [12]. For acetylcholinesterase, a protocol based on Ellman's method was used, in which 25 µL of diluted sample was mixed with 50 µL of Tris-HCl buffer (pH 8.0) and 125 µL of 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, 0.9 mM). Next, 25 µL of the enzyme was added, and the reaction mixture was incubated for 15 min at 25 • C. After the first incubation, the samples were mixed with 25 µL of acetylthiocholine iodide (ATCI, 4.5 mM) and then re-incubated for 10 min at 25 • C. The absorbance was read at 405 nm. The same protocol was applied for galantamine as positive control [12].
For the inhibition of tyrosinase, 40 µL of different concentrations from the same extract were mixed with 80 µL of PBS (with pH 6.5) and with 40 µL of enzyme in PBS, followed by an incubation for 10 min at 25 • C. After the incubation time, 40 µL of L-DOPA (10 mM, in PBS) was added to the mixture, and another incubation for 20 min at 25 • C was applied. The absorbance was read at 475 nm. The same protocol was applied for kojic acid as positive control [12].

Polyphenols Extraction
Split and intact whole fruits and separated peels were homogenized (W and P) or squeezed (J). The resulting homogenates and juices were submitted to a mild extraction method as previously reported in the Materials and Methods section (HA W , HA P and HA J ). The hydroalcoholic extraction yield ranged from 10% to 12% by dry weight in HA W and HA P accounting for the sugar content and not directly correlated to the polyphenolic content, as detailed below by HPLC analyses. The extraction yields afforded by starting from HA J showed higher ranges, between 15-16%, accounting for the more concentrated sugar content and soluble fibers of the edible part with respect to the peels represented in the whole fruits. No significant differences were shown among extracts from split or intact fruits and, on the whole, data are comparable with our previously obtained results on different pomegranate cultivars [9,13].
Considering the extraction yields related to the solid phase extraction (SPE) of anthocyanins by the hydroalcoholic extracts, yields ranging from 1.1 to 2.8% were obtained. In any case, these are only indicative because, as the anthocyanins were concentrated and Foods 2023, 12, 1908 6 of 18 made perceptible using HPLC-DAD analysis, other polyphenols and flavonoids were still present. The highest yields of SPE extracts were shown in samples obtained from split fruits (HA WA split , 2.8%; HA JA split , 1.7%) and effectively correlated with the higher anthocyanin amount found using HPLC-DAD analysis.

Colorimetric Analysis
As is well known, different pigments deeply characterize pomegranate fruit components. Anthocyanins, contained in arils, confer a brilliant red color, and yellow-brown ellagitannins are represented in both the arils and peels, contributing to or determining their color [14].
The W intact , W split , J intact , J split , HA W intact , HA W split , HA J intact , HA J split , HA P split , J S and J L samples were submitted to colorimetric CIEL*a*b* analysis. The J S and J L juices were further submitted to a shelf-life study, and the color differences were monitored over time. The CIEL*a*b* parameters are reported in Table 1. With regard to the homogenized samples (W intact and W split ) and juices (J intact , J split , J S and J L ), the L* parameter varies between 12.41 and 44.58, a* between 11.00 and 38.26 and b* between 15.29 and 31.81.
Specifically, there is no statistically significant difference ( Figure 2B) between samples W intact and W split (∆E = 5.32), and only a slight difference is observed between J intact and J split (∆E = 7.86). This mainly concerns the range between 600 and 650 nm, where J split shows a lower reflectance curve, probably due to a higher concentration of anthocyanin pigments. In fact, while the CIEL*a*b* parameters are similar in the W series (L*, 44.58 vs. 43.63; a*, 21.20 vs. 20.99; b* 17.90 vs. 19.06), in the J series, in addition to an increase in L* (12.41 rises to 19.51), a significantly higher value of a* is also observed (34.35 rises to 38.26).
In the hydroalcoholic samples (HA W intact , HA W split , HA J intact , HA J split , HA P split ), L* values ranged between 41.39 (HA W split ) and 63.96 (HA P split ), showing the highest luminance values compared to W and J before the extraction step. In this regard, as indicated from the color palette shown in Figure 2A, it is possible to observe a brighter and more brick red color for HA W samples than for W samples, with a greater difference for HA W intact (∆E = 31.65) with respect to HA W split (∆E = 21.36). Conversely, HA J samples present a lighter and more opaque reddish color than the intense mahogany coloration of the J samples, being more pronounced in HA J intact (∆E = 53.14) with respect to HA J split (∆E = 43.09). Completely different coloration is obviously presented by the HA P sample from the peels, which tends towards dark yellow.  In addition, the a* parameter drops to −1.66 in HA P split and rises to 28.86 in HA W split , whereas the b* parameter varies deeply between 0.29 (HA J intact ) and 62.59 (HA P split ). Tendentially, the a* and b* parameters are always higher in the split series, with the exception of HA W split (32.11 vs. 48.14). This difference could be related to a higher concentration of pigments (both anthocyanins and ellagitannins) in split samples than in intact ones. It is also observed that the a*/b* ratios in the HA J series are higher than in the HA W and HA P series, in relation to a higher anthocyanin content with respect to ellagitannins (see also the HPLC-DAD analysis). Conversely, the higher yellow positive b* parameter could be associated with ellagitannins [15].
The highest value of b* is found in HA P split , richer in ellagitannins as further demonstrated by the HPLC-DAD analysis. This also correlates with reflectance curves shown by Figure 2C,D. Indeed, the curves related to the hydroalcoholic extracts of the split pomegranates compared with the intact samples are lower in the region around 650 nm. In particular, there is a marked 19% decrease in reflectance for HA W split , and a slight decrease (about 2%) for HA J split , confirming the pigment's prevalence in the split samples; this behavior is evident in the redder juice samples obtained by squeezing, due to the high anthocyanin content represented in pomegranate arils [16,17]. The present data are partially comparable with those reported in the literature [13]. Figure 3A,B shows the reflectance curves related to the shelf-life study conducted on J S and J L juices kept at 40 • C for five weeks. Regarding J S , a color change is observed after the first week, and it remains constant until the third week, when a bleaching phenomenon is observed. On the contrary, around the fourth and fifth weeks, a darkening is observed, probably associated with a higher concentration of ellagitannins (see also HPLC data). A substantially different trend is registered for J L . In fact, darkening is found in the first week, followed by bleaching until the third week, and then darkening again around the fifth week.

HPLC-DAD Analysis
The different hydroalcoholic extracts obtained from pomegranate fruits of "Dente di Cavallo" and the two related commercial juices were subjected to HPLC-DAD analysis. The analyses were performed at 280 nm for the identification of phenolic acids, at 360 nm for the identification of the ellagitannin profile, mainly represented by punicalagin (α + β) and ellagic acid, and at 520 nm for the identification of the anthocyanins. Compounds were identified by external standard or by comparison with the literature [18,19]. As anthocyanins were not directly detectable from these extracts, they were analyzed after a further step of solid phase extraction, which made it possible to concentrate and quantify these pivotal compounds as well. Examples of chromatograms related to the HAW split and HAWA split samples are shown in Figure 4.

HPLC-DAD Analysis
The different hydroalcoholic extracts obtained from pomegranate fruits of "Dente di Cavallo" and the two related commercial juices were subjected to HPLC-DAD analysis. The analyses were performed at 280 nm for the identification of phenolic acids, at 360 nm for the identification of the ellagitannin profile, mainly represented by punicalagin (α + β) and ellagic acid, and at 520 nm for the identification of the anthocyanins. Compounds were identified by external standard or by comparison with the literature [18,19]. As anthocyanins were not directly detectable from these extracts, they were analyzed after a further step of solid phase extraction, which made it possible to concentrate and quantify these pivotal compounds as well. Examples of chromatograms related to the HA W split and HA WA split samples are shown in Figure 4.
The quantification of ellagitannins, reported as mg/g dry extract (Table 2), evidenced relevant differences in the range of 3-17 mg/g dry extract by juices, 21-32 mg/g dry extract by whole fruits, up to the maximum amount (77 mg/g dry extract) in the peel. Very low values were found for juices, with the exception of J S (17 mg/g by dry extract). In fact, significant differences could be seen among the three applied work-up methodologies. Juices obtained by simple pressing of the fruit (HA J , both from intact and split fruits, and J L ) show very low values of punicalagin and only in the case of J L a very slight amount of ellagic acid (<0.1 mg/g dry extract). On the contrary, in J S obtained by compression of the whole fruit, values of punicalagin and ellagic acid (17 and 1 mg/g dry extract, respectively) comparable to those recorded in the whole fruit were shown. The anthocyanin amount, yielded in relation to hydroalcoholic extracts, as well as quantified in SPE extracts, on the other hand, varied among 1 and 37 µg/g dry HA extract, reaching maxima in juice-related extracts. Anthocyanins were not detected in the J S and J L samples. The reported data agree with our previous work [9].  The quantification of ellagitannins, reported as mg/g dry extract (Table 2), e relevant differences in the range of 3-17 mg/g dry extract by juices, 21-32 mg/g d by whole fruits, up to the maximum amount (77 mg/g dry extract) in the peel. values were found for juices, with the exception of JS (17 mg/g by dry extract significant differences could be seen among the three applied work-up metho Juices obtained by simple pressing of the fruit (HAJ, both from intact and split fr JL) show very low values of punicalagin and only in the case of JL a very slight a  Further considerations were made by analyzing extracts from the intact or split fruit. As shown in Figure 5, the amount of ellagitannins, especially punicalagins, and anthocyanins appears to be higher in extracts from split fruits with respect to intact fruits. This information is very interesting, as it is related to the fact that ripening and storage can influence the matrix phytocomplex [20,21]. Such evidence could confirm what was observed in the colorimetry, as lower reflectance curves of split samples correspond to higher concentrations of ellagitannins and anthocyanins. NI: Not Identified; * expressed as punicalagin equivalents; ** expressed as µg/g dry extract of c nidin-3-rutinoside. Further considerations were made by analyzing extracts from the intact or split fru As shown in Figure 5, the amount of ellagitannins, especially punicalagins, and anthoc anins appears to be higher in extracts from split fruits with respect to intact fruits. Th information is very interesting, as it is related to the fact that ripening and storage c influence the matrix phytocomplex [20,21]. Such evidence could confirm what was o served in the colorimetry, as lower reflectance curves of split samples correspond higher concentrations of ellagitannins and anthocyanins.
The shelf-life study was conducted by storing the samples at 37 °C for 5 weeks a evaluated using both colorimetric and HPLC-DAD analyses. As shown in Figure 6, in bo JS and JL, a slight increase in ellagic acid was observed in the first two weeks, and a d crease was observed until the stabilization observed at around five weeks.
With regard to punicalagin, whereas in JL there is a sharp drop after three weeks, JS there is a slight decrease in the first weeks and then it stabilizes around four to fi weeks at lower values with respect to the t°. At the same time, punicalin, identified on in JS, increases as punicalagin decreases between the second and third week, coming ba to the initial values around five weeks. In conclusion, a decrease in punicalagin could observed, whereas punicalin and ellagic acid tend to reestablish at the initial values, in observation period of five weeks at 37 °C. Gallic acid, represented in smaller quantit comparable to those of ellagic acid, appears quite stable for the duration of the who experiment.  The shelf-life study was conducted by storing the samples at 37 • C for 5 weeks and evaluated using both colorimetric and HPLC-DAD analyses. As shown in Figure 6, in both J S and J L , a slight increase in ellagic acid was observed in the first two weeks, and a decrease was observed until the stabilization observed at around five weeks.
With regard to punicalagin, whereas in J L there is a sharp drop after three weeks, in J S there is a slight decrease in the first weeks and then it stabilizes around four to five weeks at lower values with respect to the t • . At the same time, punicalin, identified only in J S , increases as punicalagin decreases between the second and third week, coming back to the initial values around five weeks. In conclusion, a decrease in punicalagin could be observed, whereas punicalin and ellagic acid tend to reestablish at the initial values, in an observation period of five weeks at 37 • C. Gallic acid, represented in smaller quantities comparable to those of ellagic acid, appears quite stable for the duration of the whole experiment.

HS-SPME/GC-MS Analysis
GC-MS is the technique of choice to detect and identify apolar and medium-polarity metabolites arising from vegetable and food matrices [22,23]. In the present study, the HS-SPME/GC-MS analysis of split and intact HAJ and HAW samples made it possible to identify several compounds clustered according to their chemical classification (Tables 3 and  4), and these are reported in Figure 7. Aldehydes represent the prevalent class of chemicals in the HAJ samples (Figure 7), with a larger abundance in the HAJ intact, mainly due to the presence of hexanal (not detected in HAJ split), nonanal (6.4 vs. 11.2% in HAJ split and HAJ intact, respectively) and decanal (7.2 vs. 12.4% in HAJ split and HAJ intact, respectively). The HAW samples differ mainly due to the abundance of alcohol (6.8 vs. 57.9% in HAW split and HAW intact, respectively), which is completely ascribable to the presence of carvacrol in HAW intact, and the FAE distribution (24.3% in HAW split but totally absent in HAW intact). The absence of carvacrol, even in trace amounts, in all the other analyzed samples is a reasonable clue of its presence in the seeds of the pomegranate. A further comparison between the four analyzed samples reveals the following details: (i) the alkene distribution in the four analyzed samples ranges between 10.5 in HAJ intact and 20.5 in HAW split; (ii) the FAE class was detected in the HAJ split and HAW split samples (6.8 and 24.3%, respectively), but was poorly concentrated (0.9%) and absent in HAJ intact and HAW intact, respectively; (iii) methoxy phenyl oxime, a compound naturally occurring in food matrices but also recognized as a SPME fiber contaminant, was detected in all samples except for HAW intact [24,25]. Lastly, the HS-SPME/GC-MS analysis of the HAP split (Table 5 and Figure 8) pointed to aldehydes

HS-SPME/GC-MS Analysis
GC-MS is the technique of choice to detect and identify apolar and medium-polarity metabolites arising from vegetable and food matrices [22,23]. In the present study, the HS-SPME/GC-MS analysis of split and intact HA J and HA W samples made it possible to identify several compounds clustered according to their chemical classification (Tables 3 and 4), and these are reported in Figure 7. Aldehydes represent the prevalent class of chemicals in the HA J samples (Figure 7), with a larger abundance in the HA J intact , mainly due to the presence of hexanal (not detected in HA J split ), nonanal (6.4 vs. 11.2% in HA J split and HA J intact , respectively) and decanal (7.2 vs. 12.4% in HA J split and HA J intact , respectively). The HA W samples differ mainly due to the abundance of alcohol (6.8 vs. 57.9% in HA W split and HA W intact , respectively), which is completely ascribable to the presence of carvacrol in HA W intact , and the FAE distribution (24.3% in HA W split but totally absent in HA W intact ). The absence of carvacrol, even in trace amounts, in all the other analyzed samples is a reasonable clue of its presence in the seeds of the pomegranate. A further comparison between the four analyzed samples reveals the following details: (i) the alkene distribution in the four analyzed samples ranges between 10.5 in HA J intact and 20.5 in HA W split ; (ii) the FAE class was detected in the HA J split and HA W split samples (6.8 and 24.3%, respectively), but was poorly concentrated (0.9%) and absent in HA J intact and HA W intact , respectively; (iii) methoxy phenyl oxime, a compound naturally occurring in food matrices but also recognized as a SPME fiber contaminant, was detected in all samples except for HA W intact [24,25]. Lastly, the HS-SPME/GC-MS analysis of the HA P split (Table 5 and Figure 8) pointed to aldehydes as the most abundant class of compounds (66.4%), with nonanal and decanal comprising the largest part (47.3%).

Enzyme Inhibitory Activity
The HAW intact, HAW split, HAJ intact, HAJ split and HAP split samples were submitted to zymatic inhibitory activity assays, in an attempt to assess the potential to inhibit th important enzymes with implications in human physiopathology: α-glucosidase, acet cholinesterase and tyrosinase ( Table 6).
As a general trend, the HAJ intact and HAJ split samples showed the lowest inhibito activity among all, and for acetylcholinesterase and tyrosinase, we could not determ

Enzyme Inhibitory Activity
The HA W intact , HA W split , HA J intact , HA J split and HA P split samples were submitted to enzymatic inhibitory activity assays, in an attempt to assess the potential to inhibit three important enzymes with implications in human physiopathology: α-glucosidase, acetylcholinesterase and tyrosinase (Table 6). As a general trend, the HA J intact and HA J split samples showed the lowest inhibitory activity among all, and for acetylcholinesterase and tyrosinase, we could not determine any activity. In all the samples, the inhibitory activity against acetylcholinesterase and tyrosinase was weak, with values at least 100 times higher than the positive controls used. For the α-glucosidase enzyme, the only sample with activity lower than the positive control (acarbose) was HA J intact , with an IC 50 of 294.25 µg/mL. Regarding the same enzyme, the sample HA P split showed the highest inhibitory activity, with an IC 50 of 2.20 µg/mL, followed by HA W intact and HA W split . These results can also be observed in Figure 9, where the logarithmic inhibition curves show better activity than acarbose (IC 50 of 122.27 µg/mL) for all samples, with the exception of HA J intact . absorption (postprandial glycemia) from the gastrointestinal tract as adjunctive therapy of type 2 diabetes mellitus. This enzyme digests starches and carbohydrates, lowering insulin demand and sustaining a long-term release of GLP-1. Commercially available competitive and reversible inhibitors can limit the progression of diabetes but do not have any effects on pre-existing cardiovascular disease. Kam et al. (2013) [26] studied the α-glucosidase inhibitory activity of different parts of the pomegranate, showing that some phenolic species, including ellagic acid, can selectively inhibit this enzyme. Interestingly, it was also identified that the highest amount of ellagic acid and punicalagin in the HAP split sample corresponded with the highest inhibitory activity. Thus, the findings of this study highlight that the chemical composition of the phenolic content is a factor influencing the selective inhibitory effect against α-glucosidase. Furthermore, Çam and İçyer (2015) [27] found that phenolic species of pomegranate peels had an IC50 of 5.56 µg/mL for α-glucosidase, which are in line with our results. Other phenolic derivatives display inhibitory activity against this enzyme, for example, ellagitannins, ellagic acid and punicalagin from the peels [28][29][30]. The applicability of pomegranate peels as a by-product can also be further enhanced with a suitable formulation, for example, by microencapsulation [31].

Conclusions
This work allowed for a better valorization of the composition and functionality of the selected pomegranate cultivar "Dente di Cavallo", widely consumed for its excellent nutritional properties. The whole fruit, separated peels and juice produced by homemade Compounds with inhibitory activity against α-glucosidase, such as acarbose, voglibose and miglitol, have the potential to be used therapeutically in delaying glucose absorption (postprandial glycemia) from the gastrointestinal tract as adjunctive therapy of type 2 diabetes mellitus. This enzyme digests starches and carbohydrates, lowering insulin demand and sustaining a long-term release of GLP-1. Commercially available competitive and reversible inhibitors can limit the progression of diabetes but do not have any effects on pre-existing cardiovascular disease. Kam et al. (2013) [26] studied the α-glucosidase inhibitory activity of different parts of the pomegranate, showing that some phenolic species, including ellagic acid, can selectively inhibit this enzyme. Interestingly, it was also identified that the highest amount of ellagic acid and punicalagin in the HA P split sample corresponded with the highest inhibitory activity. Thus, the findings of this study highlight that the chemical composition of the phenolic content is a factor influencing the selective inhibitory effect against α-glucosidase. Furthermore, Çam andİçyer (2015) [27] found that phenolic species of pomegranate peels had an IC 50 of 5.56 µg/mL for α-glucosidase, which are in line with our results. Other phenolic derivatives display inhibitory activity against this enzyme, for example, ellagitannins, ellagic acid and punicalagin from the peels [28][29][30]. The applicability of pomegranate peels as a by-product can also be further enhanced with a suitable formulation, for example, by microencapsulation [31].

Conclusions
This work allowed for a better valorization of the composition and functionality of the selected pomegranate cultivar "Dente di Cavallo", widely consumed for its excellent nutritional properties. The whole fruit, separated peels and juice produced by homemade pressing of intact or split fruits, as well as two commercial juices, were analyzed. The two commercial juices, obtained through substantially different procedures, showed significant phytocomplex differences. The shelf-life study, conducted on color change, also demonstrated that the sample browning was directly related to the increase in ellagitannins. In intact fruits, a greater number of different volatile molecules were identified, for example, aldehydes (nonanal, decanal) in juices and peels and carvacrol in the whole fruit. Interestingly, an unneglectable amount of FAE was detected exclusively in the HA W split .
Collectively, data showed a richer chemical profile for extracts obtained from split fruit, both in terms of ellagitannins and anthocyanins. This higher bio-compound profile, especially for pomegranate juice, also leads to good health-promoting activity, most evident in α-glucosidase inhibition (IC 50 : HA J split vs. HA J intact , 110.92 vs. 294.25). Data confer an added value to this underutilized or even discarded product, suggesting that the adoption of a "zero-kilometer" approach could be carefully considered, thereby preventing its disposal and rapid deterioration and yielding a high valuable product. Finally, concerning functionality, the extracts obtained from peels, much richer in ellagitannins, showed excellent inhibitory properties against the α-glucosidase enzyme. These results, better than those exerted by acarbose, both for intact and split derived products, suggest their useful application in type 2 diabetes prevention and in the reduction in post-prandial glucose concentrations, extending their nutritional value (conventional functional foods). With respect to the current arsenal against this enzyme, the pomegranate phytocomplex contained in the juices could help avoid various side effects (diarrhea, abdominal discomfort, flatulence and bloating). Split fruits preserve this bioactivity, thus proposing themselves as valuable waste which needs to be further explored for its positive impact on an individual's health.